McNair Scholars Journal

Volume 2 | Issue 1 Article 4

Winter 1998 The ffecE ts of Intestinal Microbes on Nestling Tree Swallow (Tachycineta bicolor) Growth and Development Natasha Brock Grand Valley State University

Follow this and additional works at: http://scholarworks.gvsu.edu/mcnair

Recommended Citation Brock, Natasha (1998) "The Effects of Intestinal Microbes on Nestling Tree Swallow (Tachycineta bicolor) Growth and Development," McNair Scholars Journal: Vol. 2: Iss. 1, Article 4. Available at: http://scholarworks.gvsu.edu/mcnair/vol2/iss1/4

Copyright ©Winter 1998 by the authors. McNair Scholars Journal is reproduced electronically by ScholarWorks@GVSU. http://scholarworks.gvsu.edu/ mcnair?utm_source=scholarworks.gvsu.edu%2Fmcnair%2Fvol2%2Fiss1%2F4&utm_medium=PDF&utm_campaign=PDFCoverPages The Effects of Intestinal Microbes on Nestling Tree Swallow (Tachycineta bicolor) Growth and Development

Natasha Brock Review of the Literature used to inoculate newly hatched or juve­ McNair Scholar Microbial communities (viruses, bacteria nile birds. This inoculation insures that a and fungi) have the potential to be selec­ particular bacterial community is estab­ Patrick A. Thorpe, Ph.D. tive forces in the evolution of many lished in the developing birds. Inoculated Faculty Mentor aspects of avian biology (Hamilton and birds are less likely to harbor potentially Zuk, 1982; Hamilton, 1990; Zuk, 1991; pathogenic species, grow more rapidly; f\BSTRACT: Sheldon, 1993) but are not often consid­ and have better functioning immune Subclinical doses ofbroad-spectrum ered in studies of the dynamics of wild systems than do uninoculated birds antibiotics (Bactrim™ andClavamoxTM) bird populations. This is surprising (reviewed in Hutchenson et al., 1991.). were given tohalfof the nestlings in tree because numerous pathogenic and non­ DFM treatment has been used to restrict swallow broods as theother halfreceived pathogenic bacteria species have been iso­ or eliminate a number of undesirable bac­ a placebo. There were no differences in lated from the cloaca and pharynxes of teria species in poultry by competitive the microbial communities between the domestic and wild birds (e.g., Petrak, exclusion. Other research has shown the treated anduntreated birds on ND5or 1982; Brittingham et al., 1988; Calnek et benefits of the microbial community in a on ND19. Physical measures on ND19 al., 1991; Sheldon, 1993; Lombardo et wild bird species. Adult saliva containing showed thattreated nestlings hadsignifi­ al., 1996). Brittingham et al. (1988) sur­ a variety of microbes is required for suc­ cantly smaller tarsi andlarger mass. veyed the prevalence of selected bacteria cessful rearing of Chimney Swift in wild birds that visited winter bird feed­ (Chatetura pelagica) nestlings less than six ers in Wisconsin by using cloacal swabs days old (Kyle and Kyle, 1993). Nearly and found 44 percent of the 364 birds 100 percent of nestlings who were fed a sampled to be positive for one or more nutritionally complete food died, while of the species of bacteria tested. nearly 100 percent of those that were Pathogenic organisms can lead to fed food mixed with adult saliva were infectious diseases which can be impor­ successfully rehabilitated and released. tant sources of mortality and reduced fit­ Establishment of the microbial com­ ness in wild bird populations (Anderson munity in juvenile offspring is complex and May; 1979; Hudson and Dobson, and variable (see Savage, 1977 for a dis­ 1991). Avian cholera (Pasteurella multico­ cussion) and involves a variety of envi­ da) and botulism (Clostridium botuliniurn ronmental routes interacting with the type C) resulting in epizootics that kill specific habitat of the offspring's gastroin­ thousands of waterfowl are well known testinal tract. Individual genetic variation (Robinson and Bolen, 1989). Numerous can also affect this process (Stern et al., other incidents of disease and mortality 1990). One route involves the transmis­ caused by pathogenic bacteria are known. sion in food items that are regurgitated Such infectious diseases can result from or contaminated with adult saliva (Kyle the spread among individuals of a new or and Kyle, 1993). The microbes passed more virulent strain of bacteria, or by a from the adults are also affected by the reduction in the health of individuals mating behavior of the parents, as trans­ leading to susceptibility to pathogenic mission between adults can occur dur­ bacteria already present. ing copulation. Perek et al. (1969) Nonpathogenic bacteria also impact examined semen from 464 domestic avian health and fitness. The effects of the cockerels (Gallus domesticus) and found microbial community is especially evident that more than 36 percent of the sam­ in commercial animal husbandry where ples contained at least one bacterial the use of Direct Fed Microbials (DFM) species and that contaminated males is a common practice, particularly in infected the females with whom they domestic fowl (Hutchenson et al., 1991). copulated, demonstrating that microbes DFM represent the bacterial communities are transmitted during copulation in obtained from healthy adult birds that are birds. Communities of bacteria residing

;VSU McNairScholars Journal VOLUME 2. 1997-1998 15 in the cloacae of pair-bonded Tree given for 4 days at 24 hour intervals in an Gram positive organisms and Clavamox" Swallows (Tachycineta bicolor) were also attempt to intensify previous results. was used to target Gram negative organ­ more similar in identity than those found Physical measures related to nestling isms. Nestlings were randomly selected in non-bonded adults suggesting that growth and development were also made from a nest and alternatively given antibi­ Tree Swallows trade cloacal bacteria dur­ to detect possible effects of the alteration otic or sterile water (as a placebo). Odd ing copulation (Lombardo et al., 1996). in microbial community: numbered individuals (1,3, 5) received If the microbial communities in the antibiotics and even numbered individu­ gastrointestinal tracts of nestlings are Methods and Materials als (2, 4, 6) received the placebo .. important, if not for normal Tree swallows nest in some of the one Fresh dilutions of the antibiotics were growth and development, then drastic hundred wooden boxes mounted at the prepared daily A dose of antibiotic or alterations in these communities should Grand Valley State University campus. placebo at 33 uUg nestling weight Was have adverse effects on this process. These Tree swallows arrive in western Michigan given orally to each bird in the field using alterations would presumably be reflected in April and immediately begin looking a P200 Pipetman" micropipettor. A 3 g in a number of measures of health, for nest sites. Nestboxes were checked nestling would receive 100 uL of solution. including physical condition such as daily starting May 1, 1996, and a census Recommended dosages of Bactrim" are weight, etc. Zeller et al. (1997) have was taken to record the progress of nest doses every 12 hours at 24 mglkg and for shown that the number and diversity of building and egg laying. Eggs were Clavamox" every 12 or 24 hours at 150 the cloacal microbiota increases with age marked with indelible ink in numerical mglkg. Treatment is usually up to seven between hatching and fledging in Tree sequence as they were laid. Typically days. The actual doses received (four doses Swallows. Zeller et al. (1997) also showed females lay 1 egg per day with clutch spaced 24 hours apart, each at 48 uglg that some measures of physical condition sizes of 4-7 eggs. Fledging dates were Bactrim" and 300 uglg Clavamoxt") were significantly correlated with specific predicted based on NDI where NDI (the are substantially below recommended composition of the microbiota. Further day the first egg hatched). Nest boxes dosages for the treatment of infections. study of the relationship between bacterial were checked until all birds fledged at Recommended treatment is 7-14 dosages loads and nestling growth are justified approximately ND20. in a 7 day period; in this study; 8 doses since size at fledging is positively correlat­ To prevent early fledging, the size of were administered in a 4 day period. ed with post-fledging survival (O'Connor, the nest box hole was reduced with a 1984), and the association between bacte­ piece of tar paper on ND 18 so nestlings Physical Measures rial loads and nestling growth, survival, could not exit the box but still could On ND5 and ND12 nestlings were meas­ and fledging success has not been studied receive food from adults. The tar paper ured for flattened wing chord, bill length, in depth in wild birds. Brock et al, (1996) was removed 4-6 hours after nestlings keel length, tarsus length, head width, showed that low dosages of antibiotics did were measured on ND 19. Boxes were body length and body width. All of these change the microbial compositions of checked daily for fledging after removal of measures were also done on ND19, when treated versus untreated birds and that the tar paper. The toenails of nestlings the width of the gape was also measured. altered physical measures such as bill and were clipped to identify individuals and The length of the left and right tarsi, keel, keel length. nestlings were banded with U.S. Fish and and bill were measured with an electronic Wildlife Service aluminum bands (permit digital caliper to the nearest 0.1 mm. The Purpose of the Experiment 22299) on NDI2. The sample size for this left and right flattened wing chord, body The purpose of this experiment was to experiment included 9 nest boxes with length, and width and gape were meas­ determine if alterations in the micro biota clutch sizes of 4, 5 or 6 nestlings for a ured to the nearest 1.0 mm with a ruler of nestlings caused by antibiotic treatment total of 56 nestlings. that had a stop fixed to one end. An also cause changes in the physical meas­ Avinet spring scale was used to weigh ures of those nestlings. Since clinical treat­ Antibiotic Treatment nestlings to the nearest 0.2 g while they ment with antibiotics in birds can poten­ Subclinical doses of a mixture of Bactrim™ were briefly held in a small plastic bag. tially lead to disease from the overgrowth (a combination of sulfamethoxazole and Body condition (BC) was estimated using of non-targeted species, a subclinical dose trimethoprim) and Clavamox" (a combi­ the formula mass/keel3 (Saino and Moller, was used to determine if an alteration of nation of amoxicillin and clavulinic acid) 1994). We also calculated a Body Length! the microbiota occurred without inducing were administered to nestlings on ND 5, Body Width (LW) measurement as an disease. In this experiment doses were 6, 7 and 8. Bactrim" was used to target additional measure of condition. BC is

16 The Effects of Intestinal Microbes essentially a density measurement while After incubation all plates were then and length, and tarsus length), the treated LW is a aspect measurement. Relative counted and scores of 0, 1, 2, 3 or group had shorter extremities (wings, degrees of fluctuating asymmetry (FA) of 4 where 0 = 0 colonies, 1 = 1-10 tarsi) and shorter, larger-sized bodies bilateral traits were calculated following colonies, 2 11-100 colonies, 3 = when compared to the untreated group. Moller (1990). Fluctuating asymmetry is 101-1000 colonies, 4 = 1001- 9999 Results of the statistical analysis of the of random deviations from sym- colonies and 5 = 10,000 + colonies. Plates the physical measures, body conditions, metry in otherwise symmetrical traits that with colonies too numerous to count were and rates seen on ND12 and 19 as a of the inability of not in the analysis. Mean scores are on Tables 2, 3 and 4. The data the developing individual to cope with for individual were calculated from was analyzed using the Multivariate genetic and environmental stress (Parson, these plate scores. GLM Model. This method was used to 1990). serves as one indicator of gen- Statistical analysis.was done using the distinguish between the effects of the eral health (Moller, 1990). SPSSVersion 7.5 software package. Each treatment, the nestbox, and the effect of individual nestling was treated as an inde­ the interaction between treatment and the Cloacal Sampling and Culture pendent sample in the statistical analysis nestbox together on the nestlings. On Cloacal samples were obtained from of the relationship between microbial ND12 the nestbox had a major effect on nestlings on NDS and ND19 by inserting a loads and physical measurements for two many of the physical and condition meas­ sterile Dacron swab (5 mm long by 2 mm reasons. First, because we assumed ures for treated nestlings (Table 2). wide, Dacronstick" MW 151) into the nestlings varied genetically and second, Treatment had an effect on the left tarsus cloaca for 10 seconds. After removing the because individuals are most directly length while no effect of the combination shaft, the swab was transferred to 4 mL of effected by microbes residing in their own of treatment and nestbox was seen. On sterile thioglycollate broth. For the control, guts and not directly by the mean ND19 the nestbox alone affected the we collected field blanks by holding a ster­ microbe loads of their nestmates. Thus, mass, keel and the gape for treated ile swab in the air outside the nest box selection for resistance to pathogenic nestlings, while the treatment alone affect­ being sampled for 10 seconds and then microbes or for compatibility with mutu­ ed the tarsi and the mass. The interaction treated it as if it were a cloacal sample. alistic microbes must occur at the individ­ between treatment and the nestbox affect­ Specimens were held on ice for one hour ual level (Lombardo et al., 1996; Zeller et ed just the mass. in the field and during transport to the al., 1997). Table 3 shows the analysis of condi­ laboratory and then placed in 4°C for 30 tion measures on ND12 and 19. minutes before being plated. Immediately Results Treatment, nestbox, and the combination before plating, samples were diluted in The structure of the cloacal microbial of treatment and nestbox affected body sterile thioglycollate broth (Lombardo et communities on NDS and ND19 are condition (BC) on ND12 while the nest­ aI., 1996; Zeller et aI., 1997). shown in Figure 1. There are no differ­ box had an effect on body length/body Samples were plated on a four dif­ ences seen in the mean plate scores width (LW). On ND19 the nestbox con­ ferent selective and/or differential media between the treated and untreated groups tinued to have an effect on Be and LYV: using 3 dilutions of each sample (0.1 mL on both NDS and 19. Bacterial counts while no effect of treatment by itself or in 1 2 of 100, 10- and 10- ) in order to get were higher on ND19 than on NDS for combination with the nestbox was seen. countable plates. Samples were plated both treated and untreated birds. A simi­ Statistical analysis of the growth rates on Trypticase Soy agar (TSA) for a total lar pattern in increase was seen by Zeller on ND12 and 19 are shown in Table 4. plate count, MRS agar for lactobacilli, et al. (1997) with very low counts early in Nestbox alone on ND12 and ND19 had Sabuaroud Dextrose agar (SAB) with nestling development with counts significant effects on growth rates from chloramphenicol for fungi, and on approaching the level seen in ND19 fledg­ NDS to ND12 or ND5 to ND19. Several Levine Eosin Methylene Blue agar with lings by ND12. Many plate scores were rates were effected by the nestbox and lactose (EMB) for Gram negative organ­ zero on NDS for both treated and untreat­ most of these effects persisted until ND19. isms. All samples were plated in tripli­ ed fledglings; similar to that found by cate and incubated for 24 hours at 32°C Zeller et al. (1997). Discussion with the exception of SAB plates that Comparisons of overall conditions at The precise microbial composition of the were incubated for 36 hours. The MRS fledging (ND19) are summarized in Table gastrointestinal tract of tree swallows and plates were incubated in BBL Anaerobic 1. Of the measures taken (weight, wing its susceptibility to antibiotics is not GasPak® pouches. chord, bill length, keel length, body width known. We expect that the antibiotic

GVSU McNair ScholarsJournal VOLUME 2. 1997-1998 17 treatment does, in fact, affect the physical Nestbox effects could also have an Acknowledgments condition of nestlings as a direct result of influence on the microbe composition in This project is partof ongOing work by the microbiota alterations. Excess mortali­ individuals; due to the amount of varia­ Patrick A. Thorpe and Michael R ty in treated nestlings is not expected due tion that is seen between boxes and fami­ Lombardo of the Biology Department to the low dosages so the resulting lies (Lombardo et al., 1996), nestbox GVSU on the relationship between the condition at fledging is critical for future effects could have obscured the antibiotic internal microbiota and avian VlO-M ./1'\ 11 /"'\11"' nestling survival. The data show that effects. Teather (1996) studied nestling andevolution. Melissa Hebert, Mia combination of antibiotics used produced growth through and "" -;» y ...... "'.... "'-' ...., ..."'-' ..... Ballard, andMattCzarnowski were several effects, particularly on the tarsus. in tarsus length, bill symmetry; and feath­ invaluable field andlab assistants for Interestingly; the nestbox conditions of er symmetry He found that during the this project andothers during the1997 birds also affected the physical conditions mid to late stages of growth these meas­ season. Dr. D. McDonald provided on both ND12 and 19. This suggests an ures showed high variability due to nor­ veterinary advice particularly in the environmental effect due to nestbox con­ mal patterns of growth. Such high natural choice ofantibiotic treatment. ditions and parental behavior. variation may also have obscured the The microbiota of the internal gas­ treatment effect in our study trointestinal tract in Tree Swallows Overall, this research supports a showed no differences, by our methods, model where antibiotic .:induced alter­ between treated and untreated groups on ations in the gastrointestinal microbiota both NDS and 19. Possible reasons for alter the physical condition of the individ­ this similarity are that the doses were uals in ways that are critical for future given on NDS, 6, 7, and 8 but cloacal growth and survival. More work is needed sampling occurred only on NDS prior to to determine exactly which is the best treatment and then not until ND 19. timing for antibiotic treatment, which Recolonization of the gut may have kinds of antibiotics to use, and which occurred between the doses and the sam­ media to use in order to detect changes. pling, so that the sampling on ND 19 was Further combinations of antibiotic treat­ unable to show changes. Another possible ment regimes and sampling times should reason could be due to the types of media produce a greater effect on the microbiota used to detect changes in the microbiota. which can then be evaluated for their Since these media detect groups of bacte­ effects on growth and development. ria, changes in some specific species may not be detected. Also the media used do not detect all gastrointestinal species. The lack of effect may also be due to the fact that the antibiotics chosen did not target all of the potentially beneficial microbes so that treated and untreated individuals still had many of the same numbers and types of bacteria. There is also the possibility that the antibiotic mix­ ture chosen was correct, but the timing of doses was too early or too late to signifi­ cantly alter the critical components of the microbiota. Since microbes in the gut of nestlings have not yet been studied extensively; it is unknown what changes the microbiota undergo and therefore, what the proper time and dosages are to alter the microbiota.

18 The Effects of Intestinal Microbes Figure One. Microbial composition of treated and untreated nestlings on ND5 and ND19. Mean plate count scores and their 95 percent C.1. are shown. Individual medium codes are as used in Zeller et al. (1997). Methodology for determining the plate score is discussed in Methods and Materials.

Table 1. Conditionat Fledging. Means and standard deviations at ND19 are shown for treated and untreated nestlings. The larger measure in each category is in bold print. Physical are in gm or mm; Body Condition (Be), Body LengthIBody Width (LW) and Fluctuating Asymmetry of or Wings (FA) were calculated as described in Methods and Materials.

Table 2. Statistical analysis of Physical Measures. Physical measurements on ND12 and NP19 were compared to note the effects of treatment, nestbox, or the treatment­ nestbox interaction of both. Significant differences are indicated by p values (p

GVSU McNair ScholarsJournal VOLUME 2. 1997-1998 19 Table 3. Statistical analysis ofCondition Measures. Conditions measures for Body Condition (BC), Body LengthIBody Width (LW), and Fluctuating Asymmetry of Tarsi or Wings (FA) on ND12 and ND19 were compared the see the effects of treatment, nestbox, or the combination of both. Significant differences are indicated by p values (p

Measure

Table 4. Statistical analysis ofGrowth Rates. Growth rates from ND5 to ND12 or ND5 to ND19 were compared the see the effects of treatment, nestbox, or the combination of both. Significant differences are indicated by p values (p

20 The Effects of Intestinal References

Anderson, R. M. and R. M. May (1979). Population biology of infectious diseases, Part 1.Nature. 280: 361-367.

Brittingham, M. C., S. A. Temple, and R. M. Duncan. (1988). A survey of the prevalence of selected bacteria in wild birds.]. Wild. Diseases, 24, 299-307.

Brock, N. and P A. Thorpe. (1996). Alterations in the microbiota of (Tachycineta bicolor) and their effects on growth and development.

Calnek, B. W, H. J. Barnes, C. W Beard, W M. Read, and H. W Yoder. [Eds.]. (1991). Diseases ofpoultry, 9th Edition. Ames, Iowa: Iowa State University Press.

Hamilton, W D. (1990). Mate choice near or far. Amer. Zoology 30,341-352.

Hamilton, W D. and M. Zuk. (1982). Heritable true fitness and bright birds: a role for parasites? Science 218, 384-387.

Hudson, PJ. and A. P Dobson. (1991). The direct and indirect effects of the caecal nematode, Trichostrongylus tennius, on Red Grouse. In: Loye,]. and M. Zuk, (Eds.) Bird-parasite interactions, pp. 49-68. New York: Oxford University Press.

Hutcheson, D. P, D. C. Savage, D. S. Parker, R. D. Miles, and S. M. Bootwalla. (1991). Direct-fed microbials in animal production. A review of literature. National Feed Ingredients Association. 146 pp.

Kyle, P D., and G. Z. Kyle. (1993). An evaluation of the role of microbial flora in the salivary transfer technique for hand-rearing Chimney Swifts. Wild. Rehabilitation 8,65-71.

Lombardo, M. P, P A. Thorpe, R. Cichewicz, M. Henshaw, C. Millard, C. Steen, and T. K. Zeller. (1996). Communities of cloacal bacteria in Tree Swallow families. Condor, 98, 167-172.

Moller, A. P, K. Allander, and R. Dufva. (1990). Fitness effects of parasites on passerine birds: a review: In:]. Blondel, A. Gosler,]. D. Lebreton, and R. McCleery (Eds.), Population biology ofpasserine birds: an integrated approach, pp. 269-280. Berlin: Springer.

O'Connor, R. J. (1984). Thegrowth anddevelopment of birds. New York: Academic Press.

Parsons, P A. (1990). Fluctuating asymmetry: An epigenetic measure of stress. BioI. Review 65, 131-145.

Perek, M., M. Elian, and E. E. Heller. (1969). Bacterial flora of semen and contamination of the reproductive organs of the hen following artificial insemination. Research in Veterinary Science 10, 127-132.

Petrak, M. L. Ied.l. (1982). Diseases of cage andaviary birds. Philadelphia: Lea and Febiger.

Robinson, W L. and E. G. Bolen. (1989). Wildlife ecology andmanagement, 2nd ed. New York: Macmillan.

Saino, N. and A. P Moller. (1994). Secondary sexual characters, parasite, and testosterone in the barn swallow, Hirundo rustica. Animal Behavior 48, 1325-1333.

Savage, D. C. (1977). Microbial ecology of the gastrointestinal tract. Annals of Rev. Microbiology 31,107-133.

GVSU McNair ScholarsJournal VOLUME 2. 1997-1998 21 Sheldon, B. c. (1993). transmitted disease in birds: occurrence and c»...Trdllf-lrVnr:l1""'ir sizmncance. Phil. Trans. R. Soc. Lond. B339,491-497.

SPSS. (1996). Statistical Product and Service Solutions, Version 7.5. SPSS, Inc.

Stern, N.]., R.]. Meinersmann, N. A. Cox,]. S. and L. C. (1990). Influence of host lineage on cecal colonization by Campylobacter jejuni in chickens. Avian Dis., 34, 602-606.

Teather, K. (1996). Patterns of growth and asymmetry in nestling Tree Swallows. Journal ofAvian Biology, 27, 302-310

Zeller, T. K., Lombardo, M. P, and Thorpe, P A. (1997). Dynamics of Communities of Cloacal Microbes in Nestling Tree Swallows. In preparation.

Zuk, M. (1991). The role of parasites in sexual selection: current evidence and future directions. Advanced Study ofBehavior, 21, 39-68.

22 The Effects of Intestinal